Epigenome-wide association study of lung cancer among never smokers in two prospective cohorts in Shanghai, China.

BACKGROUND: The aetiology of lung cancer among individuals who never smoked remains elusive, despite 15% of lung cancer cases in men and 53% in women worldwide being unrelated to smoking. Epigenetic alterations, particularly DNA methylation (DNAm) changes, have emerged as potential drivers. Yet, few prospective epigenome-wide association studies (EWAS), primarily focusing on peripheral blood DNAm with limited representation of never smokers, have been conducted. METHODS: We conducted a nested case-control study of 80 never-smoking incident lung cancer cases and 83 never-smoking controls within the Shanghai Women's Health Study and Shanghai Men's Health Study. DNAm was measured in prediagnostic oral rinse samples using Illumina MethylationEPIC array. Initially, we conducted an EWAS to identify differentially methylated positions (DMPs) associated with lung cancer in the discovery sample of 101 subjects. The top 50 DMPs were further evaluated in a replication sample of 62 subjects, and results were pooled using fixed-effect meta-analysis. RESULTS: Our study identified three DMPs significantly associated with lung cancer at the epigenome-wide significance level of p<8.22×10-8. These DMPs were identified as cg09198866 (MYH9; TXN2), cg01411366 (SLC9A10) and cg12787323. Furthermore, examination of the top 1000 DMPs indicated significant enrichment in epithelial regulatory regions and their involvement in small GTPase-mediated signal transduction pathways. Additionally, GrimAge acceleration was identified as a risk factor for lung cancer (OR=1.19 per year; 95% CI 1.06 to 1.34). CONCLUSIONS: While replication in a larger sample size is necessary, our findings suggest that DNAm patterns in prediagnostic oral rinse samples could provide novel insights into the underlying mechanisms of lung cancer in never smokers.

A nested case-control study of untargeted plasma metabolomics and lung cancer among never-smoking women within the prospective Shanghai Women's Health Study.

The etiology of lung cancer in never-smokers remains elusive, despite 15% of lung cancer cases in men and 53% in women worldwide being unrelated to smoking. Here, we aimed to enhance our understanding of lung cancer pathogenesis among never-smokers using untargeted metabolomics. This nested case-control study included 395 never-smoking women who developed lung cancer and 395 matched never-smoking cancer-free women from the prospective Shanghai Women's Health Study with 15,353 metabolic features quantified in pre-diagnostic plasma using liquid chromatography high-resolution mass spectrometry. Recognizing that metabolites often correlate and seldom act independently in biological processes, we utilized a weighted correlation network analysis to agnostically construct 28 network modules of correlated metabolites. Using conditional logistic regression models, we assessed the associations for both metabolic network modules and individual metabolic features with lung cancer, accounting for multiple testing using a false discovery rate (FDR) < 0.20. We identified a network module of 121 features inversely associated with all lung cancer (p = .001, FDR = 0.028) and lung adenocarcinoma (p = .002, FDR = 0.056), where lyso-glycerophospholipids played a key role driving these associations. Another module of 440 features was inversely associated with lung adenocarcinoma (p = .014, FDR = 0.196). Individual metabolites within these network modules were enriched in biological pathways linked to oxidative stress, and energy metabolism. These pathways have been implicated in previous metabolomics studies involving populations exposed to known lung cancer risk factors such as traffic-related air pollution and polycyclic aromatic hydrocarbons. Our results suggest that untargeted plasma metabolomics could provide novel insights into the etiology and risk factors of lung cancer among never-smokers.

Genetic susceptibility to chronic diseases leads to heart failure among Europeans: the influence of leukocyte telomere length.

BACKGROUND: Genetic susceptibility to various chronic diseases has been shown to influence heart failure (HF) risk. However, the underlying biological pathways, particularly the role of leukocyte telomere length (LTL), are largely unknown. We investigated the impact of genetic susceptibility to chronic diseases and various traits on HF risk, and whether LTL mediates or modifies the pathways. METHODS: We conducted prospective cohort analyses on 404 883 European participants from the UK Biobank, including 9989 incident HF cases. Multivariable Cox regression was used to estimate associations between HF risk and 24 polygenic risk scores (PRSs) for various diseases or traits previously generated using a Bayesian approach. We assessed multiplicative interactions between the PRSs and LTL previously measured in the UK Biobank using quantitative PCR. Causal mediation analyses were conducted to estimate the proportion of the total effect of PRSs acting indirectly through LTL, an integrative marker of biological aging. RESULTS: We identified 9 PRSs associated with HF risk, including those for various cardiovascular diseases or traits, rheumatoid arthritis (P = 1.3E-04), and asthma (P = 1.8E-08). Additionally, longer LTL was strongly associated with decreased HF risk (P-trend = 1.7E-08). Notably, LTL strengthened the asthma-HF relationship significantly (P-interaction = 2.8E-03). However, LTL mediated only 1.13% (P < 0.001) of the total effect of the asthma PRS on HF risk. CONCLUSIONS: Our findings shed light onto the shared genetic susceptibility between HF risk, asthma, rheumatoid arthritis, and other traits. Longer LTL strengthened the genetic effect of asthma in the pathway to HF. These results support consideration of LTL and PRSs in HF risk prediction.

Pre-diagnostic free androgen and estradiol levels influence heart failure risk in both women and men: A prospective cohort study in the UK Biobank.

AIMS: Serum sex hormones have been linked to cardiovascular disease risk. However, their roles in the pathogenesis of heart failure (HF) in both men and women are unclear. We investigated the associations between free androgen, testosterone, and estradiol, and future risk of HF. METHODS AND RESULTS: This prospective cohort study evaluated UK Biobank participants free of prevalent cardiovascular disease and HF at baseline. Unitless free androgen, testosterone, and estradiol indices were generated using serum concentrations of total testosterone (nmol/L), estradiol (pmol/L), sex hormone binding globulin (SHBG, nmol/L), and albumin (g/L) in blood collected at enrolment. Multivariable Cox regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of incident HF in relation to quartiles (Q) of free androgen (FAI), testosterone (FTI), estradiol (FEI) indices, and potential confounders. There were 180 712 men (including 5585 HF cases with FAI and 571 HF cases with FEI), and 177 324 women (including 2858 HF cases with FAI and 314 HF cases with FEI) with complete data. Increased FAI was associated with decreased HF risk in both men (HRQ4 vs. Q1: 0.86, 95% CI 0.79-0.94, p-trendcontinuous < 0.0001) and post-menopausal women (HRQ4 vs. Q1: 0.83, 95% CI 0.73-0.95). Similar inverse associations were observed for FTI only in men (HRQ4 vs. Q1: 0.91, 95% CI 0.83-0.98). Higher FEI was significantly associated with decreased HF risk among men (HRQ4 vs. Q1: 0.76, 95% CI 0.59-0.98), but was positively associated among pre-menopausal women (HRQ4 vs. Q1: 2.16, 95% CI 1.11-4.18). CONCLUSIONS: Sex hormones potentially influence HF pathogenesis and may offer pathways for interventions.

Age at lung cancer diagnosis in females versus males who never smoke by race and ethnicity.

BACKGROUND: We characterized age at diagnosis and estimated sex differences for lung cancer and its histological subtypes among individuals who never smoke. METHODS: We analyzed the distribution of age at lung cancer diagnosis in 33,793 individuals across 8 cohort studies and two national registries from East Asia, the United States (US) and the United Kingdom (UK). Student's t-tests were used to assess the study population differences (Δ years) in age at diagnosis comparing females and males who never smoke across subgroups defined by race/ethnicity, geographic location, and histological subtypes. RESULTS: We found that among Chinese individuals diagnosed with lung cancer who never smoke, females were diagnosed with lung cancer younger than males in the Taiwan Cancer Registry (n = 29,832) (Δ years = -2.2 (95% confidence interval (CI):-2.5, -1.9), in Shanghai (n = 1049) (Δ years = -1.6 (95% CI:-2.9, -0.3), and in Sutter Health and Kaiser Permanente Hawai'i in the US (n = 82) (Δ years = -11.3 (95% CI: -17.7, -4.9). While there was a suggestion of similar patterns in African American and non-Hispanic White individuals. the estimated differences were not consistent across studies and were not statistically significant. CONCLUSIONS: We found evidence of sex differences for age at lung cancer diagnosis among individuals who never smoke.

Association between reproductive factors with lung cancer incidence and mortality: A pooled analysis of over 308,000 females in the Asia cohort consortium.

Previous studies have investigated the association between reproductive factors and lung cancer risk; however, findings have been inconsistent. In order to assess this association among Asian women, a total of 308,949 female participants from 11 prospective cohorts and four Asian countries (Japan, Korea, China, and Singapore) were included. Cox proportional hazards regression models were used to estimate the hazard ratios (HR) and 95% confidence intervals (CIs). A total of 3,119 primary lung cancer cases and 2247 lung cancer deaths were identified with a mean follow-up of 16.4 years. Parous women had a lower risk of lung cancer incidence and mortality as compared with nulliparous women, with HRs of 0.82 (95% CI = 0.70-0.96) and 0.78 (95% CI = 0.65-0.94). The protective association of parity and lung cancer incidence was greater among ever-smokers (HR = 0.66, 95% CI = 0.49-0.87) than in never-smokers (HR = 0.90, 95% CI = 0.74-1.09) (P-interaction = 0.029). Compared with age at first delivery ≤20 years, older age at first delivery (21-25, ≥26 years) was associated with a lower risk of lung cancer incidence and mortality. Women who ever used hormone replacements had a higher likelihood of developing non-small cell lung cancer (HR = 1.31, 95% CI = 1.02-1.68), compared to those who never used hormone replacements. Future studies are needed to assess the underlying mechanisms, the relationships within these female reproductive factors, and the potential changes in smoking habits over time.

Phenotypic and genetically predicted leucocyte telomere length and lung cancer risk in the prospective UK Biobank.

We investigated phenotypic leucocyte telomere length (LTL), genetically predicted LTL (gTL), and lung cancer risk among 371 890 participants, including 2829 incident cases, from the UK Biobank. Using multivariable Cox regression, we found dose-response relationships between longer phenotypic LTL (p-trendcontinuous=2.6×10-5), longer gTL predicted using a polygenic score with 130 genetic instruments (p-trendcontinuous=4.2×10-10), and overall lung cancer risk, particularly for adenocarcinoma. The associations were prominent among never smokers. Mendelian Randomization analyses supported causal associations between longer telomere length and lung cancer (HRper 1 SD gTL=1.87, 95% CI: 1.49 to 2.36, p=4.0×10-7), particularly adenocarcinoma (HRper 1 SD gTL=2.45, 95%CI: 1.69 to 3.57, p=6.5×10-6).

Lung Cancer Risk Prediction Models for Asian Ever-Smokers.

INTRODUCTION: Although lung cancer prediction models are widely used to support risk-based screening, their performance outside Western populations remains uncertain. This study aims to evaluate the performance of 11 existing risk prediction models in multiple Asian populations and to refit prediction models for Asians. METHODS: In a pooled analysis of 186,458 Asian ever-smokers from 19 prospective cohorts, we assessed calibration (expected-to-observed ratio) and discrimination (area under the receiver operating characteristic curve [AUC]) for each model. In addition, we developed the "Shanghai models" to better refine risk models for Asians on the basis of two well-characterized population-based prospective cohorts and externally validated them in other Asian cohorts. RESULTS: Among the 11 models, the Lung Cancer Death Risk Assessment Tool yielded the highest AUC (AUC [95% confidence interval (CI)] = 0.71 [0.67-0.74] for lung cancer death and 0.69 [0.67-0.72] for lung cancer incidence) and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial Model had good calibration overall (expected-to-observed ratio [95% CI] = 1.06 [0.90-1.25]). Nevertheless, these models substantially underestimated lung cancer risk among Asians who reported less than 10 smoking pack-years or stopped smoking more than or equal to 20 years ago. The Shanghai models were found to have marginal improvement overall in discrimination (AUC [95% CI] = 0.72 [0.69-0.74] for lung cancer death and 0.70 [0.67-0.72] for lung cancer incidence) but consistently outperformed the selected Western models among low-intensity smokers and long-term quitters. CONCLUSIONS: The Shanghai models had comparable performance overall to the best existing models, but they improved much in predicting the lung cancer risk of low-intensity smokers and long-term quitters in Asia.

Polygenic Risk Score, Environmental Tobacco Smoke, and Risk of Lung Adenocarcinoma in Never-Smoking Women in Taiwan.

Importance: Estimating absolute risk of lung cancer for never-smoking individuals is important to inform lung cancer screening programs. Objectives: To integrate data on environmental tobacco smoke (ETS), a known lung cancer risk factor, with a polygenic risk score (PRS) that captures overall genetic susceptibility, to estimate the absolute risk of lung adenocarcinoma (LUAD) among never-smokers in Taiwan. Design, Setting, and Participants: The analyses were conducted in never-smoking women in the Taiwan Genetic Epidemiology Study of Lung Adenocarcinoma, a case-control study. Participants were recruited between September 17, 2002, and March 30, 2011. Data analysis was performed from January 17 to July 15, 2022. Exposures: A PRS was derived using 25 genetic variants that achieved genome-wide significance (P < 5 × 10-8) in a recent genome-wide association study, and ETS was defined as never exposed, exposed at home or at work, and exposed at home and at work. Main Outcomes and Measures: The Individualized Coherent Absolute Risk Estimator software was used to estimate the lifetime absolute risk of LUAD in never-smoking women aged 40 years over a projected 40-year span among the controls by using the relative risk estimates for the PRS and ETS exposures, as well as age-specific lung cancer incidence rates for never-smokers in Taiwan. Likelihood ratio tests were conducted to assess an additive interaction between the PRS and ETS exposure. Results: Data were obtained on 1024 women with LUAD (mean [SD] age, 59.6 [11.4] years, 47.9% ever exposed to ETS at home, and 19.5% ever exposed to ETS at work) and 1024 controls (mean [SD] age, 58.9 [11.0] years, 37.0% ever exposed to ETS at home, and 14.3% ever exposed to ETS at work). The overall average lifetime 40-year absolute risk of LUAD estimated using PRS alone was 2.5% (range, 0.6%-10.3%) among women never exposed to ETS. When integrating both ETS and PRS data, the estimated absolute risk was 3.7% (range, 0.6%-14.5%) for women exposed to ETS at home or work and 5.3% (range, 1.2%-12.1%) for women exposed to ETS at home and work. A super-additive interaction between ETS and the PRS (P = 6.5 × 10-4 for interaction) was identified. Conclusions and Relevance: This study found differences in absolute risk of LUAD attributed to genetic susceptibility according to levels of ETS exposure in never-smoking women. Future studies are warranted to integrate these findings in expanded risk models for LUAD.

Associations between Longer Leukocyte Telomere Length and Increased Lung Cancer Risk among Never Smokers in Urban China.

BACKGROUND: The complex relationship between measured leukocyte telomere length (LTL), genetically predicted LTL (gTL), and carcinogenesis is exemplified by lung cancer. We previously reported associations between longer pre-diagnostic LTL, gTL, and increased lung cancer risk among European and East Asian populations. However, we had limited statistical power to examine the associations among never smokers by gender and histology. METHODS: To investigate further, we conducted nested case-control analyses on an expanded sample of never smokers from the prospective Shanghai Women's Health Studies (798 cases and 792 controls) and Shanghai Men's Health Studies (161 cases and 162 controls). We broke the case-control matching and used multivariable unconditional logistic regression models to estimate the ORs and 95% confidence intervals (CI) of incident lung cancer and adenocarcinoma (LUAD), in relation to LTL measured using quantitative PCR and gTL determined using a polygenic score. In addition, we conducted Mendelian randomization (MR) using MR-PRESSO. RESULTS: We found striking dose-response relationships between longer LTL and gTL, and increased lung cancer risk among never-smoking women (P trendLTL = 4×10-6; P trendgTL = 3×10-4). Similarly, among never-smoking men, longer measured LTL was associated with over triple the risk compared with those with the shortest (OR, 3.48; 95% CI, 1.85-6.57). The overall results were similar for LUAD among women and men. MR analyses supported causal associations with LUAD among women (OR1 SD gTL, 1.19; 95% CI, 1.03-1.37; P = 0.03). CONCLUSIONS: Longer pre-diagnostic LTL is associated with increased lung cancer risk among never smokers. IMPACT: Our findings firmly support the role of longer telomeres in lung carcinogenesis.

Mitochondrial DNA Fragmentation and Risk of Non-Hodgkin Lymphoma.

Importance: Research suggests that increased mitochondrial DNA copy number (mtDNAcn) is associated with increased risk of non-Hodgkin lymphoma (NHL); however, no studies to date have evaluated whether the mitochondrial DNA fraction with breaks (mtDNAfb) is associated with risk of NHL. Objective: To evaluate the association of mtDNAfb with NHL risk. Design, Setting, and Participants: This nested case-control study, which used prospectively collected samples as part of baseline enrollment (from 1985 through 1988) of 29 133 men who smoked for the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study conducted in southwest Finland, included 107 incident NHL cases and 107 controls (matched on date of birth ±5 years). Analyses were conducted from January to September 2022. Exposure: High-throughput real-time polymerase chain reaction assays quantifying mtDNAfb. Main Outcomes and Measures: Incident NHL cases were identified in the ATBC Study through April 30, 2002, using the Finnish Cancer Registry and the Register of Causes of Death. The mtDNAfb was quantified and categorized based on the median, tertile, and quartile distributions among controls. Odds ratios (ORs) and 95% CIs were estimated using conditional logistic regression models to assess the associations between categorized mtDNAfb and future risk of NHL, controlling for age, body mass index, number of cigarettes smoked per day, number of pack-years, and mtDNAcn. Results: A total of 29 133 men (median [IQR] age, 57.2 [52.6-62.5] years) participated in ATBC Study. Higher mtDNAfb was associated with an increased risk of NHL (median OR, 2.89; 95% CI, 1.40-5.93) in a dose-dependent manner (quartile 2 vs 1 OR, 1.24; 95% CI, 0.43-3.40; quartile 3 vs 1 OR, 3.58; 95% CI, 1.39-9.24; quartile 4 vs 1 OR, 3.42; 95% CI, 1.30- 8.99; P = .004 for trend). Conclusions and Relevance: This study's findings suggest that increased mtDNAfb is associated with an increased future risk of NHL. Additional studies are needed to confirm these findings, particularly among women and nonsmokers.

Household air pollution and epigenetic aging in Xuanwei, China.

BACKGROUND: Household air pollution (HAP) from indoor combustion of solid fuel is a global health burden linked to lung cancer. In Xuanwei, China, lung cancer rate for nonsmoking women is among the highest in the world and largely attributed to high levels of polycyclic aromatic hydrocarbons (PAHs) that are produced from combustion of smoky (bituminous) coal used for cooking and heating. Epigenetic age acceleration (EAA), a DNA methylation-based biomarker of aging, has been shown to be highly correlated with biological processes underlying the susceptibility of age-related diseases. We aim to assess the association between HAP exposure and EAA. METHODS: We analyzed data from 106 never-smoking women from Xuanwei, China. Information on fuel type was collected using a questionnaire, and validated exposure models were used to predict levels of 43 HAP constituents. Exposure clusters were identified using hierarchical clustering. EAA was derived for five epigenetic clocks defined as the residuals resulting from regressing each clock on chronological age. We used generalized estimating equations to test associations between exposure clusters derived from predicted levels of HAP exposure, ambient 5-methylchrysene (5-MC), a PAH previously found to be associated with risk of lung cancer, and EAA, while accounting for repeated-measurements and confounders. RESULTS: We observed an increase in GrimAge EAA for clusters with 31 and 33 PAHs reflecting current (ß = 0.77 y per standard deviation (SD) increase, 95 % CI:0.36,1.19) and childhood (ß = 0.92 y per SD, 95 % CI:0.40,1.45) exposure, respectively. 5-MC (ng/m3-year) was found to be associated with GrimAge EAA for current (ß = 0.15 y, 95 % CI:0.05,0.25) and childhood (ß = 0.30 y, 95 % CI:0.13,0.47) exposure. CONCLUSIONS: Our findings suggest that exposure to PAHs from indoor smoky coal combustion, particularly 5-MC, is associated with GrimAge EAA, a biomarker of mortality.

Identifying women 45 years and younger at elevated risk for endometrial hyperplasia or cancer.

OBJECTIVE: To estimate the prevalence of, and identify risk factors associated with, endometrial hyperplasia and/or cancer (EH/EC) in patients ≤45 years old undergoing endometrial sampling for abnormal uterine bleeding (AUB). METHODS: We performed a retrospective cohort study of patients 18-45 years old with AUB who underwent endometrial sampling between 2016 and 2019 within a US-based multi-hospital system using billing code queries. We used multivariable Poisson regression to identify factors associated with EH/EC and calculated prevalence stratified by these factors. We estimated predicted probabilities within combinations of characteristics in order to examine the range of risk in this population. RESULTS: Among 3175 patients, median age was 39 years (interquartile range [IQR]:35-43) and BMI was 29.7 kg/m2 (IQR: 24.2-36.9). Thirty-nine percent were non-Hispanic White, 41% non-Hispanic Black, 9% Hispanic, and 11% Asian/Other/Unknown. BMI and polycystic ovarian syndrome (PCOS) were associated with higher EH/EC risk; non-Hispanic Black race was associated with lower risk. EH/EC prevalence ranged from 2% in BMI <25 to 16% in BMI ≥50 kg/m2 (p-trend <0.001). These prevalence estimates differed by race/ethnicity with the lowest estimates in non-Hispanic Black patients (0.5% BMI <25 vs. 9% BMI ≥50) and highest in Hispanic patients (1.5% BMI <25 vs. 33% BMI ≥50). Accounting for combinations of risk factors, predicted probabilities were highest - 34-36% - among patients with PCOS, diabetes, BMI ≥50, and Hispanic or Asian/Other/Unknown race/ethnicity. CONCLUSIONS: When accounting for combinations of key risk factors, risk of EH/EC in patients ≤45 years old with AUB ranges widely; the more nuanced estimates of risk presented here could help inform clinical decision-making about endometrial sampling in this population.

Genome-wide association study of lung adenocarcinoma in East Asia and comparison with a European population.

Lung adenocarcinoma is the most common type of lung cancer. Known risk variants explain only a small fraction of lung adenocarcinoma heritability. Here, we conducted a two-stage genome-wide association study of lung adenocarcinoma of East Asian ancestry (21,658 cases and 150,676 controls; 54.5% never-smokers) and identified 12 novel susceptibility variants, bringing the total number to 28 at 25 independent loci. Transcriptome-wide association analyses together with colocalization studies using a Taiwanese lung expression quantitative trait loci dataset (n = 115) identified novel candidate genes, including FADS1 at 11q12 and ELF5 at 11p13. In a multi-ancestry meta-analysis of East Asian and European studies, four loci were identified at 2p11, 4q32, 16q23, and 18q12. At the same time, most of our findings in East Asian populations showed no evidence of association in European populations. In our studies drawn from East Asian populations, a polygenic risk score based on the 25 loci had a stronger association in never-smokers vs. individuals with a history of smoking (Pinteraction = 0.0058). These findings provide new insights into the etiology of lung adenocarcinoma in individuals from East Asian populations, which could be important in developing translational applications.

Exposure to smoky coal combustion emissions and leukocyte Alu retroelement copy number.

Household air pollution (HAP) from indoor combustion of solid fuel is a global health burden that has been linked to multiple diseases including lung cancer. In Xuanwei, China, lung cancer rate for non-smoking women is among the highest in the world and largely attributed to high levels of polycyclic aromatic hydrocarbons (PAHs) that are produced from combustion of smoky (bituminous) coal. Alu retroelements, repetitive mobile DNA sequences that can somatically multiply and promote genomic instability have been associated with risk of lung cancer and diesel engine exhaust exposure. We conducted analyses for 160 non-smoking women in an exposure assessment study in Xuanwei, China with a repeat sample from 49 subjects. Quantitative PCR was used to measure Alu repeat copy number relative to albumin gene copy number (Alu/ALB ratio). Associations between clusters derived from predicted levels of 43 HAP constituents, 5-methylchrysene (5-MC), a PAH previously associated with lung cancer in Xuanwei and was selected a priori for analysis, and Alu repeats were analyzed using generalized estimating equations. A cluster of 31 PAHs reflecting current exposure was associated with increased Alu copy number (ß:0.03 per standard deviation change; 95% confidence interval (CI):0.01,0.04; P-value = 2E-04). One compound within this cluster, 5-MC, was also associated with increased Alu copy number (P-value = 0.02). Our findings suggest that exposure to PAHs due to indoor smoky coal combustion may contribute to genomic instability. Additionally, our study provides further support for 5-MC as a prominent carcinogenic component of smoky coal emissions. Further studies are needed to replicate our findings.

Alterations to biomarkers related to long-term exposure to diesel exhaust at concentrations below occupational exposure limits in the European Union and the USA.

BACKGROUND: We previously found that occupational exposure to diesel engine exhaust (DEE) was associated with alterations to 19 biomarkers that potentially reflect the mechanisms of carcinogenesis. Whether DEE is associated with biological alterations at concentrations under existing or recommended occupational exposure limits (OELs) is unclear. METHODS: In a cross-sectional study of 54 factory workers exposed long-term to DEE and 55 unexposed controls, we reanalysed the 19 previously identified biomarkers. Multivariable linear regression was used to compare biomarker levels between DEE-exposed versus unexposed subjects and to assess elemental carbon (EC) exposure-response relationships, adjusted for age and smoking status. We analysed each biomarker at EC concentrations below the US Mine Safety and Health Administration (MSHA) OEL (<106 µg/m3), below the European Union (EU) OEL (<50 µg/m3) and below the American Conference of Governmental Industrial Hygienists (ACGIH) recommendation (<20 µg/m3). RESULTS: Below the MSHA OEL, 17 biomarkers were altered between DEE-exposed workers and unexposed controls. Below the EU OEL, DEE-exposed workers had elevated lymphocytes (p=9E-03, false discovery rate (FDR)=0.04), CD4+ count (p=0.02, FDR=0.05), CD8+ count (p=5E-03, FDR=0.03) and miR-92a-3p (p=0.02, FDR=0.05), and nasal turbinate gene expression (first principal component: p=1E-06, FDR=2E-05), as well as decreased C-reactive protein (p=0.02, FDR=0.05), macrophage inflammatory protein-1ß (p=0.04, FDR=0.09), miR-423-3p (p=0.04, FDR=0.09) and miR-122-5p (p=2E-03, FDR=0.02). Even at EC concentrations under the ACGIH recommendation, we found some evidence of exposure-response relationships for miR-423-3p (ptrend=0.01, FDR=0.19) and gene expression (ptrend=0.02, FDR=0.19). CONCLUSIONS: DEE exposure under existing or recommended OELs may be associated with biomarkers reflective of cancer-related processes, including inflammatory/immune response.

Methylated polycyclic aromatic hydrocarbons from household coal use across the life course and risk of lung cancer in a large cohort of 42,420 subjects in Xuanwei, China.

BACKGROUND: We previously showed that exposure to 5-methylchrysene (5MC) and other methylated polycyclic aromatic hydrocarbons (PAHs) best explains lung cancer risks in a case-control study among non-smoking women using smoky coal in China. Time-related factors (e.g., age at exposure) and non-linear relations were not explored. OBJECTIVE: We investigated the relation between coal-derived air pollutants and lung cancer mortality using data from a large retrospective cohort. METHODS: Participants were smoky (bituminous) or smokeless (anthracite) coal users from a cohort of 42,420 subjects from four communes in XuanWei. Follow-up was from 1976 to 2011, during which 4,827 deaths from lung-cancer occurred. Exposures were predicted for 43 different pollutants. Exposure clusters were identified using hierarchical clustering. Cox regression was used to estimate exposure-response relations for 5MC, while effect modification by age at exposure was investigated for cluster prototypes. A Bayesian penalized multi-pollutant model was fitted on a nested case-control sample, with more restricted models fitted to investigate non-linear exposure-response relations. RESULTS: We confirmed the strong exposure-response relation for 5MC (Hazard Ratio [95% Confidence Interval] = 2.5 [2.4, 2.6] per standard-deviation (SD)). We identified four pollutant clusters, with all but two PAHs in a single cluster. Exposure to PAHs in the large cluster was associated with a higher lung cancer mortality rate (HR [95%CI] = 2.4 [2.2, 2.6] per SD), while exposure accrued before 18 years of age appeared more important than adulthood exposures. Results from the multi-pollutant model identified anthanthrene (ANT) and benzo(a)chrysene (BaC) as risk factors. 5MC remained strongly associated with lung cancer in models that included ANT and BaC and also benzo(a)pyrene (BaP). CONCLUSION: We confirmed the link between PAH exposures and lung cancer in smoky coal users and found exposures before age 18 to be especially important. We found some evidence for the carcinogen 5MC and non-carcinogens ANT and BaC.

Occupational exposure to diesel engine exhaust and serum levels of microRNAs in a cross-sectional molecular epidemiology study in China.

Diesel engine exhaust (DEE) is an established lung carcinogen, but the biological mechanisms of diesel-induced lung carcinogenesis are not well understood. MicroRNAs (miRNAs) are small noncoding RNAs that play a potentially important role in regulating gene expression related to lung cancer. We conducted a cross-sectional molecular epidemiology study to evaluate whether serum levels of miRNAs are altered in healthy workers occupationally exposed to DEE compared to unexposed controls. We conducted a two-stage study, first measuring 405 miRNAs in a pilot study of six DEE-exposed workers exposed and six controls. In the second stage, 44 selected miRNAs were measured using the Fireplex circulating miRNA assay that profiles miRNAs directly from biofluids of 45 workers exposed to a range of DEE (Elemental Carbon (EC), median, range: 47.7, 6.1-79.7 µg/m3 ) and 46 controls. The relationship between exposure to DEE and EC with miRNA levels was analyzed using linear regression adjusted for potential confounders. Serum levels of four miRNAs were significantly lower (miR-191-5p, miR-93-5p, miR-423-3p, miR-122-5p) and one miRNA was significantly higher (miR-92a-3p) in DEE exposed workers compared to controls. Of these miRNAs, miR-191-5p (ptrend  = .001, FDR = 0.04) and miR-93-5p (ptrend  = .009, FDR = 0.18) showed evidence of an inverse exposure-response with increasing EC levels. Our findings suggest that occupational exposure to DEE may affect circulating miRNAs implicated in biological processes related to carcinogenesis, including immune function.

Association between diesel exhaust exposure and mitochondrial DNA methylation.

OBJECTIVES: Diesel exhaust is an established human carcinogen, however the mechanisms by which it leads to cancer development are not fully understood. Mitochondrial dysfunction is an established contributor to carcinogenesis. Recent studies have improved our understanding of the role played by epigenetic modifications in the mitochondrial genome on tumorigenesis. In this study, we aim to evaluate the association between diesel engine exhaust (DEE) exposure with mitochondrial DNA (mtDNA) methylation levels in workers exposed to DEE. METHODS: The study population consisted of 53 male workers employed at a diesel engine manufacturing facility in Northern China who were routinely exposed to diesel exhaust in their occupational setting, as well as 55 unexposed male control workers from other unrelated factories in the same geographic area. Exposure to DEE, elemental carbon, organic carbon, and particulate matter (PM2.5) were assessed. mtDNA methylation for CpG sites (CpGs) from seven mitochondrial genes (D-Loop, MT-RNR1, MT-CO2, MT-CO3, MT-ATP6, MT-ATP8, MT-ND5) was measured in blood samples. Linear regression models were used to estimate the associations between DEE, elemental carbon, organic carbon and PM2.5 exposures with mtDNA methylation levels, adjusting for potential confounders. RESULTS: DEE exposure was associated with decreased MT-ATP6 (difference = -35.6%, P-value = 0.019) and MT-ATP8 methylation (difference = -30%, P-value = 0.029) compared to unexposed controls. Exposures to elemental carbon, organic carbon, and PM2.5 were also significantly and inversely associated with methylation in MT-ATP6 and MT-ATP8 genes (all P-values < 0.05). CONCLUSIONS: Our findings suggest that DEE exposure perturbs mtDNA methylation, which may be of importance for tumorigenesis.

Exposure to diesel engine exhaust and alterations to the Cys34/Lys525 adductome of human serum albumin.

We investigated whether exposure to carcinogenic diesel engine exhaust (DEE) was associated with altered adduct levels in human serum albumin (HSA) residues. Nano-liquid chromatography-high resolution mass spectrometry (nLC-HRMS) was used to measure adducts of Cys34 and Lys525 residues in plasma samples from 54 diesel engine factory workers and 55 unexposed controls. An untargeted adductomics and bioinformatics pipeline was used to find signatures of Cys34/Lys525 adductome modifications. To identify adducts that were altered between DEE-exposed and unexposed participants, we used an ensemble feature selection approach that ranks and combines findings from linear regression and penalized logistic regression, then aggregates the important findings with those determined by random forest. We detected 40 Cys34 and 9 Lys525 adducts. Among these findings, we found evidence that 6 Cys34 adducts were altered between DEE-exposed and unexposed participants (i.e., 841.75, 851.76, 856.10, 860.77, 870.43, and 913.45). These adducts were biologically related to antioxidant activity.